Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels (h channels) mediate the hyperpolarization-activated current (Ih) and are important for regulating excitability in CA1 pyramidal neurons of the hippocampus. Because localization governs h channel function in the homeostasis of neuronal excitability, and h channel dysfunction has been implicated in temporal lobe epilepsy, our longterm goal is to understand the unknown trafficking and targeting mechanism of h channels in the hippocampus, h channels of CA1 pyramidal neurons as well as neocortical layer V neurons exhibit a striking distal dendritic enrichment (DDE), shown to be critical for control of cellular excitability and synchrony. One molecular candidate implicated in h channel trafficking is the tetratricopeptide repeat-containing (TPR) Rab8b interacting protein (TRIPSb). TRIPSb is the only known interactor of h channels to demonstrate pyramidal neuron DDE in both the hippocampus and cortex mimicking that of HCN1 and 2. Additionally, TRIPSb has been shown to regulate Ih density in oocytes. TRIPSb interacts with h channels via its conserved C-terminal TPR domains in a manner homologous to the peroxisomal import receptor proteins. However, the N-terminal region of TRIPSb shares no homology with any known protein and its function is unknown. Our preliminary data indicate the N-terminus is highly alternatively spliced in a developmentally regulated manner, whereby specific isoforms are upregulated concurrent with DDE onset. Notably, alternative splicing alters the presence of cellular sorting signals thought to be important in trafficking. Because of this considerable but indirect evidence associating TRIPSb with h channel trafficking, we reason that TRIPSb plays an important role in the establishment and/or maintenance of h channel DDE in the hippocampus. To determine TRIPSb's role in hippocampal DDE, we propose the following specific aims: 1) Determine whether TRIPSb in general, and the N-terminus of TRIPSb in particular, are required for h channel DDE in CA1 pyramidal neurons. We will use lentiviral delivery of shRNA and dominant negative TRIPSb constructs in both slice culture and the living animal to determine the effect on h channel DDE in the hippocampus in vivo. 2) Determine the role of TRIPSb N-terminal alternative splicing in h channel trafficking and DDE. We will use single-cell RT-PCR, biotinylation, and viral delivery of TRIPSb splice isoforms to determine how TRIPSb splicing impacts h channel surface trafficking and DDE. PUBLIC HEALTH RELEVANCE: epilepsy remains a major lifelong burden. By understanding how the brain regulates excitability, we will gain insight into the causes of epilepsy and mechanisms for targeted treatment.